Re: [Histonet] EGFP IHC in floating brain sections

From:

"Mark Tarango"

I just realized that you suggested going to 1:1000 for that antibody but you
said "increasing the concentration." Going from 1:500 to 1:100 is diluting
it out further... anyway I do think that should help, if it doest... again
maybe an avidin/biotin blocking kit would help.
Mark
On Wed, Sep 17, 2008 at 10:23 PM, Mark Tarango wrote:
> Hi Caroline,
>
> I didn't see an avidin/biotin block but noticed that your donkey anti-goat
> has biotin as its label. Do you think that a blocking step might clear up
> the background on the ones w/ the strong signal (because of endogenous
> biotin in the brain getting stained)?
>
> Or maybe it would be easier to clean up the staining if you tried something
> in the range of 1:600- 1:1000 for that biotin labeled seconary.
>
> Just some thoughts,
>
> Mark
>
> On Wed, Sep 17, 2008 at 11:12 AM, Caroline Bass wrote:
>
>> Hello,
>>
>> Sorry if this is a double post but I didn't see my first one in the
>> digest.
>>
>> Iım hoping someone could help me troubleshoot my staining protocol. I am
>> staining to EGFP in microglia (produced by a virus I injected in the
>> brain).
>> Hereıs my general protocol:
>>
>> Deactive endogenous peroxidases (0.1M PB + 20% methanol + titon-x100 (80
>> ul
>> in 40 ml total) + 0.3% h2O2) for 15 min.
>> Wash 3X15min in 0.1MPB+0.3%titon
>> Incubate in 0.1MPB+0.3%triton+1%normal donkey serum for 1 hr
>> Incubate with primary (goat anti-GFP from Rockland) overnight at 4 degrees
>> (1:1000)
>> Wash 3x15 min 0.1M PB + 0.3% triton
>> Incubate in with secondary (Jackson Donkey anti-goat, biotin labeled)
>> overnight at 4 degrees (1:500)
>> Wash 3x15 min in 0.1M PB
>>
>> Use ABC kit and vector SG for substrate (canıt remember how long I let it
>> develop, but it was within a couple of minutes).
>>
>> So, the bottom line is that I did a test section and I thought it had a
>> lot
>> of background, so I incubated the other sections for less time. However,
>> when I took a closer look, the sections with little background had no
>> microglial signal, while the one with high background had a very clear and
>> noticeable signal in the place it should be. So, the question is how to
>> cut
>> back on the noise. Would increasing the secondary concentration to 1:1000
>> help? Iım following a protocol someone else came up with, but it seems
>> strange that the secondary is at a higher concentration than the primary.
>>
>> Thanks!
>>
>> Caroline
>>
>>
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>
>
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